Antibiotic purification



United States Patent 2,897,234 Patented July 28, 1959 ANTIBIOTICPURIFICATION Bernard Heinemann, East Syracuse, and Irving R.

Hooper, Fayetteville, N.Y., assignors to Bristol Laboratories Inc.,Syracuse, N.Y., a corporation of New ork No Drawing. Application April'18-, 1956 Serial No. 578,872

1 Claim. (Cl. 260-659)" This invention is concerned with novel anduseful methods for the purification of tetracycline and relates moreparticularly to methods for its recovery in purified form from solidmaterials or solutions of such materials containing both tetracyclineand chlortetracycline.

This application is a continuation-in-partof our prior co-pendingapplication Serial No. 432,388, filed May 26, 1954-, now'abandoned.

The useful broad spectrum antibiotic, tetracycline, has been obtained bycatalytic deschlorinationof chlortetracycline according to Conover, US.Patent 2,699,054.

Tetracycline, often admixed with chlortetracycline, is also obtained byfermentation of Streptomyces aureofaciens or szreptomyces viridifaciense.g. according to US. Patents 2,712,517, 2,734,018 and 2,739,924 andaccording to vBohonoset al., Antibiotics Annual, 1953-4, pages 49-55;The usual processes for the recovery of tetracycline from a fermentationbroth which contains both tetracycline and chlortetracycline yield solidproducts containing these two antibiotics in substantially the sameratio as they occur in the fermentation broth. Their chemical andphysical properties are so similar as to make it extremely difficult toremove the undesired chlortetracycline by a method suitable forcommercial use as opposed to purely laboratory methods such as the useof Craig countercurrent distribution. Such purification is, of course,essential before tetracycline can be marketed for therapeutic use.

The object of the present invention is to provide improved methods ofremoving contaminating amounts of chlortetracycline from tetracyclinewhich are suitable for commercial use.

In accordance with the present invention there is provided the processof recovering substantially chlortetracycline-free tetracycline from asolid mixture of tetracycline and chlortetracycline which comprisesslurrying said solid mixture in a minimal amount of aqueous acid ofabout pH 2.5, using no less than 0.1 milliliter of aqueous acid permilligram of chlortetracycline, until substantially all of saidchlortetracycline has dissolved, and then collecting the purified,solid, undissolved tetracycline.

After the completion of a fermentation to produce a broth containing amixture of tetracycline and chlortetracycline, tetracycline is recoveredfrom the broth, for example, by filtering to remove the mycelium(preferably at about pH 2.0), stirring the broth (preferably at about pH8.5) with butanol or methyl isobutyl ketone, separating the solventlayer containing the tetracyclincs, concentrating it to small volume bydistillation, and simply cooling, or mixing it with a liquid loweraliphatic hydrocarbon, e.g., Skellysolve C, to precipitate solid, mixedtetracyclines.

In another process the crude tetracycline is recovered from thefermentation broth by an alkaline precipitation. Fundamentally, thefermentation broth is adjusted to an alkaline pH in the range of 8 to 11inclusive and the precipitated mixture of mycelium (mat) andtetracycline is collected by filtration. As a variant of this process,the

fermentation broth is acidified to less than pH 3, filtered to removethe mycelium, andthe filtrate is treated as above, that is it isadjusted to an alkaline pH inthe range of 8 to 11 inclusive and theprecipitate of crude tetracycline is collected by filtration. Any watersoluble alkali of sufiicient strength is used, ammonia or sodiumhydroxide are preferred. The pH is adjusted to the range of 8 to 11inclusive; an alkaline pH in the range of 9 to 10 is preferred. Theprecipitate of tetracycline, with or without accompanying mycelium, isbrought into solution for further manipulation as desired according toany of the procedures disclosed herein by solution in' aqueous acidbelow pH 3 or in aqueous alkali above pH 11. Examples of such aqueousacids include water-soluble acids furnishing the desired pI-I, e.g.hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid. Thesesame acids are effective in lowering the pH of fermentation broth belowpH 3, and preferably to about pH 2.0-2.5, to permit separation oftetracycline, which is-in solution as an acid addition salt, from thesolid mycelium by filtration before use of the alkaline precipitationdescribed above.

Tetracycline is converted to tetracycline salts by dissolving the basein dry acetone or n-propanol and adding the anhydrous acid, i.e.,hydrogen chloride gas, sulfuric acid, sulfuric acid in dry acetone,phosphoric acid, tartaric acid, citric acid, nitric acid in dry methylisobutyl ketone, and collecting by filtration the salt whichprecipitates, e.g., tetracycline hydrochloride or the sulfate,phosphate, tartrate, citrate, etc.

In another procedure for the recovery of a mixture of tetracycline andchlortetracycline from a fermentation broth, there is added to thefermentation broth 0.1% CaCl and the broth is adjusted to about pH8.5-9.0 with caustic, e.g. soda or potash, or ammonia and then extractedwith n-butanol, using /2 volume for batchwise extraction or volume formultiple stage extraction. The mixture is filtered and the butanol phaseis separated if batchwise procedure is used. The tetracycline-containingbutanol is concentrated to V of the original broth volume bydistillation in vacuo. The butanol concentrate and any insolublestherein are extracted with three successive equal volumes of wateradjusted to pH 2.5 with hydrochloric acid. These aqueous extracts arecombined, filtered and concentrated by distillation in vacuo to 4 brothvolume. To this aqueous concentrate is added 1% calcium chloride (w./v.)and the pH is adjusted to about 7.8-8.5 by the addition of ammoniumhydroxide. The resulting precipitate of solid crude calcium tetracyclineis collected by filtration and dried with an acetone wash and air orvacuum drying.

A very simple, inexpensive and efficient method of isolating crude solidtetracycline from large volumes of fermentation broth is furnished bythe following procedure. A fermentation broth containing at least 250meg/m1. tetracycline, and preferably 1000 meg/ml. tetracycline, or more,is adjusted to an acid pH and the mycelium is removed by filtration. Thefiltered broth is then adjusted to pH 8 or higher by the addition ofcaustic, e.g. soda, or ammonia. Crude, active, solid tetracyclineprecipitates as the free base or calcium salt, contaminated with calciumphosphates and other impurities, and is collected by filtration.

Tetracycline is efficiently purified of contaminating amounts ofchlortetracycline, when such are present, by slurrying in aqueous acidof substantially pH 2.5, which dissolves only the chlortetracycline. Thepreferred acid is hydrochloric acid; other acids, e.g. sulfuric acid,phosphoric acid, may be used, however, provided the requisite pH isobtained. Tetracycline base or salt is slurried in hydrochloric acidsuflicient to provide a pH of 2.5 or, as is preferred, impuretetracycline base or salt, e.g. hydrochloride, is dissolved in aqueousacid at pH 1.5

or less and sufficient base, e.g. ammonium hydroxide, sodium hydroxide,is added to provide a pH of 2.5. The amount of tetracycline used is nota limiting factor; in general it is convenient to use 50 to 100 mgms.tetracycline base or salt per milliliter of water acidified to pH 2.5with hydrochloric acid. The pH is a critical factor; substantially pH2.5 is preferred although any pH in the range 2.0-3.5 is useful. Theamount of chlortetracycline present as an impurity in tetracycline to bepurified by this procedure, should not exceed 9 mgms./ml. of acidifiedwater and preferably should be less than mgm./ml.; this limit is easilyobserved by increasing when necessary the amount of acid water used forslurrying. Such solids, e.g. tetracycline hydrochloride andchlortetracycline hydrochloride, in this purification are thus mixed,i.e., slurried, in water acidified with hydrochloric acid tosubstantially pH 2.5 for a considerable period of time, e.g. sixteenhours, sufiicient to permit the chlortetracycline to dissolve. This isdetermined by simple test. The purified, solid tetracycline base whichremains undissolved is then collected by filtration.

Further understanding of the present invention is provided by thefollowing examples which will serve for purposes of illustration, butnot of limitation.

Example I Tetracycline hydrochloride (10 g.) assaying 830 mcg./mgm.tetracycline and 171 mcg./mgm. chlortetracycline (83% tetracycline) bythe differential ultra-violet absorption method and containing about 10%chlortetracycline as determined by paper strip chromatography, wasslurried for sixteen hours in 200 ml. water adjusted to about pH 2.5with hydrochloric acid. The solid, purified tetracycline base was thencollected by filtration, dried, found to weigh 8.0 grams, to assay 901mcg/mgm. tetracycline and 39 mcg./mgm. chlortetracycline (96%tetracycline) by the differential ultra-violet absorption method and tocontain 95-97% tetracycline as determined by paper-strip chromatography.The recovery of tetracycline was 87%.

Example 11 An experiment similar to that of Example I was conductedunder nitrogen and gave a 92% recovery of tetracycline base containingfrom zero to three percent chlortetracycline, potency 929 mcg./mgm.

Quantitative analysis of mixtures of tetracycline and chlortetracyclineis carried out according to the procedures in the literature, e.g.Antibiotic Annual, 19531954, pages 54 and 55 and pages 8187; Minieri etal., U.S. Patent 2,734,018.

We claim:

The process of recovering substantially chlortetracycline-freetetracycline from a solid mixture of tetracycline and chlortetracyclinewhich comprises slurrying said solid mixture in a minimal amount ofaqueous mineral acid of about pH 2.5, using no less than 0.1 milliliterof aqueous acid per milligram of chlortetracycline, until substantiallyall of said chlortetracycline has dissolved and for at least aboutsixteen hours, and then collecting the purified, solid, undissolvedtetracycline.

References Cited in the file of this patent FOREIGN PATENTS 583/55 Unionof South Africa Sept. 14, 1955

